Vibrio cholerae causes the disease cholera and is a natural inhabitant of aquatic environments. Seasonal cholera outbreaks occur where the disease is endemic and can spread worldwide. V. cholerae's ability to form biofilms (i.e., matrix-enclosed, surface-associated communities) is crucial for its survival in aquatic habitats between epidemics and is advantageous for host-to-host transmission during epidemics. The objective of this proposal is to improve our understanding of biofilm matrix components, the mechanisms and regulation of biofilm formation, and their importance in the biology of V. cholerae. In Aim 1, we will focus on understanding how biofilm matrix proteins RbmA, RbmC and Bap1 function. We will determine the molecular basis of RbmA and VPS interactions and determine molecular determinants and consequences of RbmA proteolysis. We will test whether RbmC and Bap1 bind to VPS and identify regions important for their function. We will also determine how these proteins contribute to the mechanical properties of biofilms, as well as the location of each matrix component by super resolution microscopy. In Aim 2, we will determine the mechanism used by the regulatory proteins, VpsR and VpsT, in biofilm formation. We will identify sensor histidine kinases that phosphorylate VpsR, and determine phosphorylation dynamics of VpsR during biofilm development. We will also identify direct targets of VpsT and VpsR during biofilm formation and ascertain the contribution of VpsR and VpsT regulon members in V. cholerae transmission and dissemination into the environment. Clarifying the mechanisms of biofilm formation in V. cholerae will prove useful for the development of future strategies for controlling cholera epidemics, and facilitate identification of novel drug targets for combating the pathogen during infection.